Double stranded RNA has been shown to be a powerful agent for interfering with gene expression in a number of organisms, including C. elegans and Drosophila, as well as plants (Bernstein et al, RNA 7:1509–2151 (2001), McManus et al, Nat. Rev. Genet. 3:737–747 (2992), Hutvagner et al, Curr. Opin. Genet. Dev. 12:225–232 (2002), Zamore, Nat. Struct. Biol. 8:746–750 (2001) Tuschl et al, Genes Dev. 13:3191–3197 (1999)). Early problems in silencing mammalian genes with double stranded RNA arose because the mammalian immune system destroys cells that contain double stranded RNA, through mechanisms such as the interferon response, evolved as defense against invading RNA viruses (Clarke et al, RNA 1:7–20 (1995)). It has been demonstrated, however, that very short RNA fragments (e.g., 20–23 nt in length), designated small interfering RNA (siRNA), are able to escape the immune response. Thus introduced siRNAs can function well as gene silencing agents in mammalian cells (Elbashir et al, Nature 411:494–498 (2001), Elbashir et al, Genes Dev. 15:188–200 (2001), Paddison et al, Genes Dev. 16:948–958 (2002), Wianny et al, Nat. Cell Biol. 2:70–75 (2000)).
As it is presently understood, RNAi involves a multi-step process. Double stranded RNAs are cleaved by the endonuclease Dicer to generate 21–23 nucleotide fragments (siRNA). The siRNA duplex is resolved into 2 single stranded RNAs, one strand being incorporated into a protein-containing complex where it functions as guide RNA to direct cleavage of the target RNA (Schwarz et al, Mol. Cell. 10:537–548 (2002), Zamore et al, Cell 101:25–33 (2000)), thus silencing a specific genetic message (see also Zeng et al, Proc. Natl. Acad. Sci. 100:9779 (2003)).
Anti-sense DNA has also been widely used to inhibit gene expression (Roth et al, Annu. Rev. Biomed. Eng. 1:265–297 (1999)). Once inside the cell, anti-sense oligonucleotides (ASO) recognize, then bind tightly to complementary mRNA, thus preventing the mRNA from interacting with the protein translation machinery of the cell.
It has been demonstrated that inhibition of Syk kinase expression by Syk kinase mRNA ASO dramatically diminishes Fcγ receptor signaling (Matsuda et al, Molec. Biol. of the Cell 7:1095–1106 (1996)), and that Syk kinase mRNA ASO introduced by aerosol into rat lungs protects against Fcγ receptor-induced lung inflammation (Stenton et al, J. Immunol. 169:1028–1036 (2002)).
At least in certain systems, siRNA is more potent and reliable than ASO as an inhibitor of gene expression. The present invention results from studies designed to test the efficacy of siRNA as an inhibitor of Syk kinase expression.